1. Field of the Invention
The invention relates to a DNA hybridization incubator, of a type usefully employed to perform blot DNA hybridizations and stringency washes for Southern, Northern, and Western blot procedures.
2. Description of the Related
The study of gene structure, function, and expression involves the extensive use of DNA and RNA hybridization techniques, in which these nucleic acids are hybridized to suitable radioactive nucleic acid probes.
In such techniques, nucleic acids to be studied may be separated according to size by electrophoresis in a gel medium, e.g., agarose gel, and the resulting separated nucleic acid is immobilized by transfer to a suitable immobilization medium such as nitrocellulose paper or a polymeric membrane. The separated blots then may be washed in a buffer medium containing the radioactive probe, e.g., a .sup.32 P-labeled DNA probe, which selectively hybridizes to the nucleic acid molecules complementary in nucleotide sequence to the probe.
The hybridization of DNA and RNA probes with nucleic acid on filter media involves Southern and Northern blotting techniques. In a related technique applicable to the study of proteins, termed Western blotting, protein blots are incubated with antibodies.
In these various hybridization procedures, depending on reagent (buffer) compositions employed, and the similarity of the probe and target molecules, the temperatures employed may variously range from about ambient temperature to about 70.degree. C. Temperature is usefully employed as a process variable in altering the hybridization stringency. At the lower end of the aforementioned temperature range, heterologous probes are effectively used, while at the high end, target sequences which are substantially identical to the nucleotide sequence of the probe are required for the hybridization.
Typically, nucleic acid and protein hybridizations are carried out in a closed container in a constant temperature environment for extended periods of time, e.g., 10-18 hours.
The prior art has utilized various heating systems and arrangements for hybridization, including: (i) plastic bags with water baths, in which the plastic bags containing the blots and buffer medium are introduced to a constant temperature shaking water bath, with the shaking serving to produce temperature uniformity and to effect intimate contacting of the blots with the buffer medium; (ii) conventional ovens, with agitation of the buffer/blot medium contained therein; and (iii) so-called hybridization incubators, in which a rotisserie or rack is mounted in a temperature-controlled convection heating enclosure, and is driven in continual movement during the heating operation.
In relative merit, hybridization incubators have major advantages over plastic bag/water bath systems and conventional ovens with ancillary stirring or agitation of the blot/buffer medium, and have come into increasingly widespread usage in recent years. Various commercialized hybridization incubators are described below.
The BELLCO.RTM. AutoBlot.TM. Hybridization Oven, commercially available from BELLCO Glass, Inc. (Vineland, N.J.) is a hybridization apparatus designed for incubation/washing experiments requiring the use of membranes, including Northern, Southern, Dot, Slot, Colony, and Western blot techniques. This oven features a rotisserie oriented with its axis of rotation parallel to the front door of the oven. The rotisserie accommodates a multiplicity of incubator bottles thereon, and is removable from the oven cavity. A slip clutch is provided in the rotisserie drive mechanism to permit removal of the rotisserie from the oven even when the rotisserie drive mechanism is actuated. Forced air circulation is employed to ensure temperature uniformity, and minimize the occurrence of "hot spots" in the oven chamber.
The GFL.RTM. Hybridisation Incubator 7601 (distributed by Man-Tech Associates, Inc. (Buffalo, N.Y.)) is an incubator device utilizing forced air circulation means and a rotating rack including two large round discs with holes around their edges to enable insertion of hybridization tubes into the rack. Two wheels with spring clips mounted between these two discs hold the hybridization tubes. The clip wheel on one side can be slid along the axle, and additional clip wheels can be added to accommodate shorter hybridization tubes and to increase the incubator unit's capacity.
The Robbins Scientific.RTM. Model 310 Hybridization Incubator features a digital proportional-integral-derivative controller which measures and controls the incubator chamber temperature. A separate temperature sensor output is provided for continuously recording chamber temperature of the unit by an optional recorder. A tridirectional rotator comprising a ferris wheel clamp assembly is disposed in the chamber, on which multiple glass tubes are mounted for rotation, with the tubes being resultingly rotated about the main central axis of the rotator, as well as rotation about the glass tube axis, and with the tubes also being imparted with a back-and-forth movement, so that in Western blot procedures the blot proteins are exposed to a continuously moving thin film of antisera.
Other hybridization incubator apparatuses which are commercially available include: the Techne HB-1 Hybridization Incubator (Daigger Scientific Products, Manassas, Va.); the Hybridizer.TM. 600 Hybridization Oven (Stratagene Corporation, San Francisco, Calif.); and the Hybaid Mini Hybridization System (National Labnet Company, Woodbridge, N.J.).
In all of the above-described incubator structures, the heating (oven) chamber is essentially a conventionally configured gravity- or forced-convection oven with a rotisserie or rotator structure mounted or placed in a chamber of square or rectangular cross-section along each of its three major (x,y,z) axes.
These hybridization incubators, although a major advance over the aforementioned plastic bag and water bath systems, and an improvement over use of agitated containers in ovens per se, nonetheless are characterized by the inability to consistently and reliably achieve and maintain a selected temperature uniformity (throughout the entire volume of the incubator chamber), stability (over the duration of the hybridization or wash procedure), and accuracy (relative to the selected chamber temperature) within a tolerance of about .+-.0.5.degree. C. or less.
This is a particular shortcoming in many applications in which the temperature must be varied according to a rigorous time-temperature schedule in order to satisfactorily carry out the procedure, with only a low tolerance in the temperature variations and deviations being permitted.
Accordingly, it is an object of the present invention to provide an improved hybridization incubator which is optimally configured to achieve better temperature uniformity, stability, and accuracy than has heretofore been possible with the hybridization incubator apparatuses of the prior art.
It is another object of the present invention to provide a hybridization incubator which is of an improved configuration with respect to its safety and maintenance characteristics, over hybridization incubators of the prior art.
Other objects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.